The Raw Milk Microbiota from Semi-Subsistence Farms Characteristics by NGS Analysis Method
Total Page:16
File Type:pdf, Size:1020Kb
molecules Article The Raw Milk Microbiota from Semi-Subsistence Farms Characteristics by NGS Analysis Method Bartosz Hornik 1 , Jakub Czarny 1 , Justyna Staninska-Pi˛eta 2 , Łukasz Wolko 3 , Paweł Cyplik 4 and Agnieszka Piotrowska-Cyplik 2,* 1 Institute of Forensic Genetics, Al. Mickiewicza 3/4, 85-071 Bydgoszcz, Poland; b.hornik@igs.org.pl (B.H.); pubjc@igs.org.pl (J.C.) 2 Department of Food Technology of Plant Origin, Poznan University of Life Sciences, Wojska Polskiego 31, 60-624 Pozna´n,Poland; justyna.staninska@up.poznan.pl 3 Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Dojazd 11, 60-632 Pozna´n,Poland; lukasz.wolko@up.poznan.pl 4 Department Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-627 Pozna´n,Poland; pawel.cyplik@up.poznan.pl * Correspondence: agnieszka.piotrowska-cyplik@up.poznan.pl; Tel.: +48-618487284 Abstract: The aim of this study was to analyze the microbiome of raw milk obtained from three semi- subsistence farms (A, B, and C) located in the Kuyavian-Pomeranian Voivodeship in Poland. The composition of drinking milk was assessed on the basis of 16S rRNA gene sequencing using the Ion Torrent platform. Based on the conducted research, significant changes in the composition of the milk microbiome were found depending on its place of origin. Bacteria belonging to the Bacillus (17.0%), Citation: Hornik, B.; Czarny, J.; Corynebacterium (12.0%) and Escherichia-Shigella (11.0%) genera were dominant in the milk collected Staninska-Pi˛eta,J.; Wolko, Ł.; from farm A. In the case of the milk from farm B, the dominant bacteria belonged to the Acinetobacter Cyplik, P.; Piotrowska-Cyplik, A. The genus (21.0%), whereas in the sample from farm C, Escherichia-Shigella (24.8%) and Bacillus (10.3%) Raw Milk Microbiota from Semi- dominated the microbiome. An analysis was performed using the PICRUSt tool (Phylogenetic Subsistence Farms Characteristics by Investigation of Communities by Reconstruction of Unobserved States) in order to generate a profile NGS Analysis Method. Molecules 2021, 26, 5029. https://doi.org/ of genes responsible for bacterial metabolism. The conducted analysis confirmed the diversity of the 10.3390/molecules26165029 profile of genes responsible for bacterial metabolism in all the tested samples. On the other hand, simultaneous analysis of six KEGG Orthologs (KO), which participated in beta-lactam resistance Academic Editors: Antonio- responsible for antibiotic resistance of bacteria, demonstrated that there is no significant relationship José Trujillo, Radmila Pavlovic, between the predicted occurrence of these orthologs and the place of existence of microorganisms. Luca Chiesa, Sara Panseri and Therefore, it can be supposed that bacterial resistance to beta-lactam antibiotics occurs regardless of Emanuela Zanardi the environmental niche, and that the antibiotic resistance maintained in the population is a factor that shapes the functional structure of the microbial consortia. Received: 23 June 2021 Accepted: 16 August 2021 Keywords: antibiotic resistance; raw milk microbiome; farm; next-generation sequencing Published: 19 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Milk and its products play an important role in human nutrition in many cultures. It is a source of protein, vitamins and many minerals, which makes it an excellent environment for the growth and development of bacteria [1,2]. Raw cow milk is one of the most diverse raw materials in terms of microbiology, which directly affects the quality and price of manufactured products and the company’s financial results [3]. The colonization of milk Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. by microorganisms is a significant threat that has a negative impact not only on the quality This article is an open access article and durability of products, but also on human health [4]. In order to achieve a positive distributed under the terms and effect of the health properties of milk on the human body, appropriate hygienic conditions conditions of the Creative Commons should be maintained not only during collection, but also during its transport to dairy Attribution (CC BY) license (https:// plants, processing and preservation. creativecommons.org/licenses/by/ Milk, in addition to its endogenous microbiota, enables the development of microor- 4.0/). ganisms which may originate from the surface of animals, as well as from the environment Molecules 2021, 26, 5029. https://doi.org/10.3390/molecules26165029 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 5029 2 of 12 in which the livestock lives [4]. The contamination of milk with bacteria mainly results from poorly cleaned and poorly disinfected milking equipment, lack of hygiene during milking or handling of the milk, and bacteria present in the barn. Moreover, the presence of environmental pathogens such as Escherichia coli, Klebsiella spp., Streptococcus dysgalactiae and Streptococcus uberis may contribute to the development of mastitis in dairy cattle, which is an “occupational disease” of high-yielding dairy cows [5,6]. The disease is caused by bacteria and other microorganisms that enter the teats. The appearance of this disease in high-yielding dairy cows is the cause of huge economic losses [7]. There are two forms of mastitis: clinical and subclinical, which is a latent form. It is estimated that up to 50% of cows may suffer from subclinical mastitis. Unfortunately, in the subclinical form, there are also economic losses caused by the reduced milk yield of cows; hence, the correct diagnosis and implementation of an effective treatment process are very important [8]. The treatment of mastitis in cows, despite the implementation of prophylactic pro- grams and new methods of therapy, is still mainly based on the administration of antibiotics. It is estimated that animal production in most countries sometimes accounts for as much as 80% of the total consumption of antibiotics [9]. Unfortunately, their presence in food has negative health and economic consequences for humans [10]. One of the greatest threats to public and global health associated with the use of antibiotics is the increase in antibiotic resistance of bacteria [11]. It is assumed that the main cause of this phenomenon is their excessive use, and the scale of abuse in this area makes them the main cause of the growth and spread of antibiotic-resistant bacteria and resistance genes in the environment [12]. Antibiotics used to treat and prevent bacterial infections in animals may also contribute to the formation of drug-resistant bacterial strains in the human body [13]. It can be said with certainty that the process of managing antibiotic resistance has progressed considerably, meaning that it is leading the world into the post-antibiotic era. Unfortunately, the alarming forecasts from scientists have become completely realistic, and harmless bacterial infections that have been successfully treated in the past appear to be deadly today [14]. Among the antibiotics, more than 60% of the intramammary preparations used contain β-lactams (penicillins, cephalosporins), which is confirmed by the results of the presence of antibiotics in milk. One of the main reasons for the insensitivity to antibiotics is the ability of bacteria to produce β-lactamase, an enzyme that neutralizes the action of, for example, penicillins and cephalosporins [12]. On the one hand, the subthreshold concentrations of antibiotics strongly influence the selection of resistant strains, and they contribute to the formation of morphologically changed bacteria [15,16]. In addition, the environment in which antibiotics are present favors the transfer of mobile genetic elements by hori- zontal gene transfer, which leads to the dissemination of resistance genes even between phylogenetically distant bacteria [17]. Due to the above-mentioned reasons, the assessment of the composition and direction of development of the microbiota present in raw milk has a significant impact not only on the composition and quality of milk, but above all on the quality of dairy products [18]. The microorganisms present in these products may affect human health. They constitute a reservoir of genes that determine the antibiotic resistance of bacteria, which can be permanently transferred to the microbiome of the human gastrointestinal tract [19]. On this basis, sequence analysis of the hypervariable regions of the 16S rRNA gene using the Ion Torrent platform (Life Technologies, Carlsbad, CA, USA) was used to assess the microbiome of milk from semi-subsistence farms. Then, a functional analysis of the milk microbiome was carried out based on the PICRUSt tool in order to determine the potential of the microbiome as a carrier of genes that determine the resistance of bacteria to β-lactam antibiotics. Molecules 2021, 26, x 3 of 13 Molecules 2021, 26, 5029 3 of 12 2. Results and Discussion 2.1. Taxonomic Analysis of the Milk Microbiome Milk is is an an excellent excellent environment environment for for the the development development of of microorganisms microorganisms responsi- respon- blesible for for the the specific specific properties properties of ofmany many dairy dairy products products as well as well as microbes as microbes which which are un- are desirableundesirable for fortechnological technological and andhealth health reasons reasons (review (review by Quigley by Quigley